Pain Exposure and Brain Connectivity in Preterm Infants

Key Points Question Are there sex-specific associations among early-life pain exposure, neonatal brain network maturation, and neurodevelopmental outcomes in preterm infants? Findings In this prospective cohort study of 150 preterm infants, sex-specific associations were found between early-life pain exposure and maturation of neonatal brain structural connectivity, with a greater association with pain seen in female infants. Decreased brain structural connectivity was associated with poorer 18-month neurodevelopmental outcomes. Meaning In this study, early-life pain exposure was associated with slower maturation of structural brain networks, particularly in female infants; clinical trials of neonatal analgesic strategies should consider this sex-specific vulnerability to early-life pain.


Invasive procedures
Each attempt at the following procedures was included, consistent with previous work. 1

Definitions of clinical data
Postnatal culture positive infection was defined as any positive blood or cerebrospinal culture, necrotizing enterocolitis (NEC) as stage 2 or higher, retinopathy of prematurity (ROP) as requiring treatment, chronic lung disease (CLD) as requiring supplemental oxygen at 36 weeks' PMA, and major surgeries as requiring laparotomy, thoracotomy, ostomy, extracorporeal membrane oxygenation or surgery involving the central nervous system.Extreme prematurity was defined as birth gestational age  28 weeks.Analgesic exposure (morphine, fentanyl, midazolam) was categorized based on duration of exposure as no exposure, exposure for short durations (7 days or less) or long durations (longer than 7 days).

DTI Preprocessing and tractography
We excluded scans of 29 patients with extensive brain abnormalities including periventricular hemorrhagic infarction, stroke, brain malformations and severe ventriculomegaly which would have impacted tractography.Data from 11 scans were excluded following visual quality control checks; 141 early-life scans and 131 TEA scans were included in subsequent analyses.
Diffusion images were corrected for head motion (including intervolume motion correction, slice-to-volume motion correction) 2 , susceptibility 3 , and eddy currents using FSL eddy. 4 Diffusion parameters were estimated in each voxel using FSL BEDPOSTX. 5e performed probabilistic tractography, allowing fractional anisotropy values to influence the termination of tracts, as implemented in FSL PROBTRACKX2. 6,7FA is influenced by axonal density, packing, orientation, and myelination, and is typically used as a measure of white matter maturation.Mean fractional anisotropy (FA), a measure of overall connectivity strength, was calculated for each scan using a brain mask.

Network Construction
To define anatomical regions of interest, we warped neonatal atlases 8,9 to each subject's diffusion image using ANTs 10 , first from the atlas to each subject's T2 image, followed by nonlinear transformations from each T2 image to the corresponding diffusion image.Combining these transformations yielded 92 region labels in each subject's diffusion space excluding primarily white-matter regions, cerebrospinal fluid, and the brainstem; these were manually reviewed for accuracy of transformations.
All measures were computed using the Brain Connectivity Toolbox for Python. 11To examine changes in functional segregation and integration, we constructed weighted structural networks for each subject.The 92 anatomical regions were used as nodes.
To construct edges, we applied a proportional thresholding approach, in which the ratio of actual connections to possible connections is set to a fixed density for all subjects.The weights of the edges connecting two nodes were initially set to the number of tractography streamlines connecting pairs of nodes.The weakest edges were then set to zero, denoting non-connections, until the specified density was reached.Because network properties vary with density, we constructed networks over a range of densities from 0.01 to 0.1 in steps of 0.01.At the density of 0.1, at least 90% of subjects had unthresholded network densities greater than the threshold.
To ensure that our findings were not tied to a specific network density, we used an area under the curve (AUC) approach.Network metrics were computed at each density threshold, and the final value used in analysis was the following modified area under the curve: Here, i is the metric value at the  th network density, i.The sum represents the area under the metric-density curve.With the coefficient on the sum, the expression represents the mean of the metric over the range of densities, approximated using the trapezoidal rule.This definition returns the metric to its scale on a single network, facilitating its interpretation and comparison with values in other networks.
To measure network integration, we computed global efficiency for weighted networks.To measure network segregation, we computed the network local efficiency by averaging local efficiency across the network.To measure the extent to which networks exhibited the small world property, we calculated small-worldness.
Regional connectivity strength was quantified by the average number of tractography streamlines connecting each region to every other region without thresholding.To measure thalamocortical connection strength, we computed the average number of tractography streamlines connecting the left and right thalamus components to all cortical regions.We used analogous procedures to measure cortico-striatal, and thalamo-striatal connection strengths.
Sensitivity analyses were performed after removing infants with moderate-severe WMI.Early-life pain by PMA at scan interaction remained significantly associated with global (eTable 3; interaction p=0.002) and local (p=0.005)efficiency in females only, adjusting for PMA at scan, mean FA and moderate-severe WMI.
Structural connectivity and neurodevelopmental outcomes: Regional connectivity In GEE of regional network connectivity and neurodevelopmental outcomes, greater connection strengths in the left and right putamen were associated with higher Cognitive scores, adjusting for PMA at scan, extreme prematurity, and maternal education, and correcting for multiple comparisons (eTable 8).Greater connection strength in the left caudate was also associated with higher Language scores (eTable 8).There were no significant associations between regional connection strength and motor outcomes in any region.Maternal education was associated with neurodevelopment in these models; children born to mothers who completed postgraduate level education had higher Cognitive, Motor and Language scores compared to mothers whose highest level of education was elementary/high school.Abbreviations: CI = confidence interval; Coef = coefficient.
Umbilical arterial or venous catheter insertion Peripheral arterial line stab Peripheral intravenous line insertion Peripherally inserted central catheter insertion Venous blood draw Heel poke Intramuscular or subcutaneous injection Chest tube insertion Pleural tap Penrose drain insertion Paracentesis Intubation Suprapubic tap Omaya reservoir tap Circumcision PICC dressing change or removal Intravenous infiltration and extravasation Penrose drain removal Ostomy change Endotracheal tube retaping or suction Nasopharygeal or oropharyngeal suction Orogastric tube insertion and removal Nasogastric/Nasojejunal tube insertion and removal Dressing change X-ray Eye exam Invasive swab Chest compressions Ultrasound Intraventricular hemorrhage was scored according to the Papile score.
Univariable generalized estimating equations of associations between earlylife painful exposures and global efficiency.Similar associations were observed with local efficiency.There were no significant relationships with small worldness.Sensitivity analyses with generalized estimating equations of early-life invasive procedures x PMA at scan interaction and measures of network segregation and integration stratified by sex after excluding patients with moderate-severe WMI.Generalized estimating equations of early-life invasive procedures x postmenstrual age at scan interaction and corticostriatal, thalamocortical and thalamostriatal connection strength, stratifying by sex.Generalized estimating equations of early-life invasive procedures and regional connectivity.Q values indicate significance level after FDR correction.Simple model adjusting for postmentstrual age at MRI; complex model adjusting for postmenstrual age at MRI and moderate-severe WMI.
Clinical characteristics for infants who did and did not complete neurodevelopmental assessments.Univariable generalized estimating equations of associations between clinical variables and Bayley-3 scores.Generalized estimating equations of associations of measures of network segregation and integration with Bayley-3 scores at 18 months, adjusting for postmenstrual age at scan, global fractional anisotropy, extreme prematurity, maternal education, and clinical confounders Models adjusting for: birth gestational age, days of mechanical ventilation, culture positive infections, and major surgery. *